ADP-ribosylation factors (ARFs) are about 20 kDa proteins that act as GTP- dependent allosteric activators of cholera toxin. Using inosine-containing degenerate oligonucleotide primers directed to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids and intervening sequences. Amplification products, which differed in molecular weight because of differences in intron sizes, were separated by agarose gel electrophoresis. One amplified DNA contained no introns, and had a sequence different from but related to those of known ARFs. Based on this apparently unique sequence, selective oligonucleotide probes were prepared and used to isolate clone (psi)ARF 4, a putative ARF pseudogene, from a human genomic library in lambda phage EMBL3. Reverse transcription-PCR, using primers in the 5'- and 3' untranslated regions, was then used to clone from human poly(A)+ RNA a CDNA containing the entire coding sequence corresponding to the expressed homolog of (psi)ARF 4, and referred to as human ARF 4. It appears that (psi)ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs significantly from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat poly(A)+ RNA revealed a single 1.8 kb mRNA, which was clearly distinguishable from the ARF 1 1.9 kb mRNA. The polymerase chain reaction provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance. Current investigations focus on the diversity of the ARF multigene family, the relationship between ARFs and other multigene families coding for guanine-nucleotide binding proteins, and evolutionary relations of ARF genes across species.